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doi: 10.1242/10.1242/jcs.00137

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RNA trafficking and stabilization elements associate with multiple brain proteins

Mark Snee*, Grahame J. Kidd1, Trent P. Munro2 and Ross Smith{ddagger}

Department of Biochemistry and Molecular Biology, The University of Queensland, Qld 4072, Australia
1 Department of Neurosciences NC30, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland OH 44195, USA
2 Wellcome/CR UK Institute, Tennis Court Rd, Cambridge CB2 1QR, UK
* Present address: Institute for Cellular and Molecular Biology, University of Texas, 2500 Speedway, Austin, TX, USA



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  		Fig. 1. A2RE11, zipcode and AURE bind different groups of proteins. Coomassie-blue-stained SDS/polyacrylamide gel showing rat brain proteins that bound to magnetic particles bearing immobilized MBP mRNA trafficking sequence (A2RE11), ß-actin mRNA 5' zipcode (5'ZIP), the AU-rich response element (AURE) or a non-specific RNA sequence (NS1). This figure was created from an image of a single gel by deletion of every second track, which contained irrelevant samples. The positions of standard proteins are shown on the left, with molecular masses in kDa.

 


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  		Fig. 2. Western blots confirm specificity of binding to zipcode and A2RE11. (A) 21-day-old rat brain proteins were used in pull-down experiments with magnetic particles bearing NS1, A2RE11 or 5' zipcode. The RNA-bound proteins were eluted, run on SDS/polyacrylamide gels and electroblotted. The blots were incubated with the indicated primary antibodies and protein bands detected with alkaline phosphatase-conjugated secondary antibody. The molecular masses in kDa are indicated on the left. (B) NS1-, AURE- and 5'-zipcode-binding proteins detected with anti-KSRP (C2742). (C) Western blots developed with chicken antibody to CRD-BP, which binds ZBP-1. On the left, proteins from embryonic (E15) and 21 day postnatal (P21) rat brains. No 68 kDa protein was observed in pull-down experiments with the A2RE or 5' zipcode and P21 chicken (centre) or P21 rat (right) brain protein. This blot was overdeveloped to show the weak band. (D) Western blot of rat brain proteins bound to NS1, A2RE11 and 5' zipcode detected with antibodies to conventional kinesin heavy chain. The positions of standard proteins are shown on the left, with molecular masses in kDa.

 


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  		Fig. 3. The 3' zipcode binds a subset of the 5'-zipcode-binding proteins isolated from rat brain protein extracts. (A) Coomassie-blue-stained SDS/polyacrylamide gel showing rat brain proteins that bound to magnetic particles bearing immobilized ß-actin mRNA 5' zipcode (5'ZIP) or the 3' zipcode (3'ZIP). The bands identified by Edman sequencing are marked on the right. In this experiment, as in others we have performed, the level of hnRNP L was higher than in the experiment shown in Fig. 1. The positions of standard proteins are shown on the left, with molecular masses in kDa. (B) Western blotting of proteins from pull-down experiments performed with immobilized A2RE11, 5'ZIP and 3'ZIP showed that hnRNP A2 does not bind the 5' zipcode or 3' zipcode. The blot was developed with hnRNP A2 antibody as the primary antibody. Protein bands were detected with an alkaline-phosphatase-conjugated secondary antibody.

 


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  		Fig. 4. Zipcode- and A2RE-binding proteins are present in rat cortical neurons and glial cells. Sections of cortex of 15-day-old rats were stained for KSRP (A,B red) FBP (C,D red), hnRNP A2 (E,F red) and hnRNPs A1 (G red), B1 (H red), and A3 (I red) by immunoperoxidase (A,C,E) and immunofluorescence methods (B,D,F-I). Most neurons (arrows in A,C, E) and all white matter glia (arrowheads in A,C,E) were labeled for KSRP, FBP and hnRNP A2. Confocal microscopy of sections double-labeled with oligodendrocyte markers CC1 (green in B,D, which show regions of white matter) and CNP (green in F-I, which show cortical grey matter) detected KSRP, FBP, hnRNP A2 and hnRNP A3 in these cells (arrows). hnRNP A1 was not detected in oligodendrocytes (G, arrowhead), and many were also B1-negative (H, arrowhead). wm and gm: white and grey matter. Bars, 100 µm for (A,C,E) and 25 µm for other images.

 


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  		Fig. 5. KSRP and hnRNP A2 are concentrated in separate regions in the nuclei of cultured oligodendrocytes. KSRP (A and red in C; C2742 antibody) and hnRNP A2 (B and green in C; chicken antibody) are present at high levels in the nuclei with concentration of these proteins in regions that appear to contain primarily one protein or the other but not both. D-G show a mitotic cell in which KSRP (D, red in G) is concentrated in areas that largely exclude hnRNP A2 (E, green in G), which is associated with the dividing chromatin (F, blue DAPI staining in G). Bars, A-C, 2 µm, D-G, 5 µm.

 


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  		Fig. 6. Extranuclear hnRNP A2 and KSRP are differentially localized. (A) A cultured oligodendrocyte showing the presence of KSRP (C2742 antibody, red) and hnRNP A2 (green) in the nucleus and processes. In the cell body the relative level of hnRNP A2 is higher. The two asterisks mark what appear to be adjacent cells. (B) A magnified view of a process of the cell from multiple merged optical sections. Distinct populations of granules containing KSRP (arrowheads) or hnRNP A2 (arrows) are evident in the oligodendrocyte processes. (C) A histogram showing the fluorescence ratios [red/(red + green); see Materials and Methods] of granules in four cells (excluding the cell shown in A) double-labeled for hnRNP A2 (green) and KSRP (red). Bars, A, 10 µm, B, 2.5 µm.

 

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